Antithrombin III is a glycoprotein and plays a key role in maintaining the fluidity of the circulating blood. It is my aim to study the molecular structure of this inhibitor, both physically and chemically, and the results obtained herein may further our understanding of the physiological function of this macromolecule at the molecular level. Antithrombin III is purified from bovine plasma mainly by heparin-sepharose affinity chromatography and DEAE-cellulose gradient chromatography. This new isolation procedure is a relatively simple one and the yield is very high. The homogeneity of this preparation is evaluated by ultracentrifugation, dodecyl sulfate gel electrophoresis and thin-layer gel electrofocusing. Concomitant with this ultracentrifugal investigation, the molecular weight of this antienzyme is determined and this value is substantiated by independent methods of dodecyl sulfate gel electrophoresis and gel filtration in 6M guanidinium chloride. The total quantitative amino acid composition of this glycoprotein is determined by the automatic amino acid analyzer and the carboxyl-terminal amino acid is elucidated by the method of hydrazinolysis. Each neutral and amino sugar is analyzed quantitatively by gas chromatography or silica gel thin-layer chromatography. The stoichiometry of the binding of heparin to this inhibitor is investigated by sephadex gel filtration. BIBLIOGRAPHIC REFERENCE: Yue, R.H., Starr, T. and Gertler, M.M. Isolation and Partial Characterization of Bovine Antithrombin III Federation Proc. 35, 322 (1975).